The objectives of this application are to intensify ongoing research on the physiology of synaptic cotransmission in sympathetic ganglia and to learn molecular and biological methods that will be applied in new studies of neuronal differentiation. The P.I. is an assistant professor of Physiology and is in the 05 year of independent research with RO1 support. An excellent collegial environment and adequate support services exist within the local University community. The propose plan will insure continued development of the P.I.'s research career. It will limit growth of non-research related activities and provide resources to pursue new methodologies, experiments and collaborations. The experiments will utilize a combination of electrophysiological, anatomical and biochemical methods in isolated preparations and in primary cell cultures. The new methods to be learned include nucleic acid isolation,Northern analysis and in situ hybridization. The experimental goals of the proposal are to determine the integrative function and the developmental origins of synapses that utilize slowly acting peptidergic co-transmitters in addition to the classical transmitters acetylcholine (ACH) and epinephrine (EPI). The roles of 4 neuropeptides, luteinizing hormone releasing hormone (LHRH), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP) and substance P (SP), will be analyzed in the sympathetic system of bullfrog. Although these peptides are co-expressed with other transmitters in many regions of the peripheral and central nervous system, the functional dynamics and ontogeny of synaptic co-transmission is largely unknown. Bullfrog sympathetic ganglia express a diversity of synaptic mechanisms and yet they are accessible to analysis at the cellular and molecular levels. In the lumbar ganglia, there exist 3 subclasses of sympathetic neurons that can be distinguished by their functions, electrophysiological properties, connectivity with the spinal cord and with peripheral targets, and by their expression of muscarinic cholinergic receptors and neuropeptides. In the vasomotor sympathetic C system, LHRH and ACH are co-released by preganglionic neurons and elicit 3 postsynaptic potentials in a subset of ganglion cells that co-express EPI and NPY-like immunoreactivity. The first specific aim of this proposal is to test the hypothesis that co-transmission at ganglionic and end-organ synapses in this circuit enhances the dynamic range of vascular contractions that can be elicited by different patterns of preganglionic stimulation. The second specific aim is to test the hypothesis that CGRP is co-released with ACH by preganglionic B neurons and produces trophic effects and an excitatory postsynaptic potential in B neurons. The third goal is to establish the timetable during development in tadpoles for cell-specific expression of neuropeptide genes and their products. Using tissue culture, the final goal of the project will be to test the hypothesis that expression of NPY is controlled by molecules in the extracellular environment and/or by cellular interactions, and that such factors also control the electrophysiological differentiation of vasomotor neurons.